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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Published in a peer-reviewed journal

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Percutaneous absorption through skin samples from rats, rabbits and humans was assessed using 14C-radiolabeled test material.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexane-1,3-diol
EC Number:
202-377-9
EC Name:
2-ethylhexane-1,3-diol
Cas Number:
94-96-2
Molecular formula:
C8H18O2
IUPAC Name:
2-ethylhexane-1,3-diol
Test material form:
liquid: viscous
Details on test material:
no data on purity
2-ethyl-1,3-hexane diol (EHD)
Radiolabelling:
yes
Remarks:
[14C]-labeled EHD

Test animals

Species:
other: Human, rat and rabbit
Strain:
other: Fischer 344 rats, New Zealand White rabbit
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Fischer 344 rats, 10-12 weeks old, obtained from Harlan Sprague Dawley (Indianapolis, IN, USA). Food and water was available ad libitum. New Zealand white rabbits, male and female, were 12-13 weeks old when obtained from Hazleton Research Products Inc. (Denver, PA, USA). Agway PRO-LAB Certified rabbit diet (Agway Inc., St. Mary's, OH, USA) was available ad libitum; water was from a municpal source, and available ad libitum. All animals were acclimated for 3 days prior to obtaining skin samples. Animals were housed in stainless steel cages. Temperature and humidity were monitored continuously for a 12 h light/dark schedule.
Human skin samples were obtained from mammoplasty patients in the University of Pittsburgh Hospital system.

Administration / exposure

Type of coverage:
other: occlusive and open
Vehicle:
water
Duration of exposure:
6 h
Doses:
undiluted and 3% aqueous solution
No. of animals per group:
3 animals/sex/group.
3 human skin samples per dose group
Control animals:
yes
Details on study design:
Human skin samples were obtained from mammoplasty patients. Upon removal, the specimens were placed into minimum essential media (MEM/d-valine: Eagles medium with Earle's salts (Eagle , 1959) and 25 mM Hepes buffer (Gibco); with penicillin/streptomycin), until prepared and placed in the skin penetration chamber. Human samples were used within 1-2 h after excision from donor. Skin discs were prepared by a modification of the method of Kao, et al., 1983, for full-thickness excised skin preparations. Prior to removal of the skin samples, the rats were given an anesthetic overdose of Metofane and the rabbits were injected with Beuthanasia solution. The fur was clipped from the dorsal trunk in the thoracic region. Skin (6x6 cm) was removed and placed in MEM solution; human skin was received in MEM and processed similarly to animal skin. The serosal surface of the skin pieces was gently scraped with a spatula to remove fat and loose connective tissue. Two 1-inch discs from each animal skin, and six 1-inch discs from human skin were placed in a Petri dish containing MEM prior to positioning in the chamber.
Details on in vitro test system (if applicable):
[14C]-labeled EHD was applied as either an undiluted liquid dose under an occlusive covering (to simulate in vivo dosing conditions) or as a 3% aqueous solution in an amount which covered the entire dosing surface of the skin disc in an infinite dose approach for the 6-h period. The opening of the chamber was either sealed to prevent evaporative losses, or was open. A target radioactivity of 5-10 µCi/skin preparation and target concentrations of 36 and 25 mg/cm2 of skin surface were applied to male and female skin preparations, respectively. Dosing solutions were analyzed by liquid scintillation spectometry.

The apparatus and techniques were modifications of Holland, et al., 1984, as published by Frantz et al., 1990. Flow rate of perfusion was 2.6 ml/h for at least 30 min prior to application of the test chemical. Undiluted EHD was applied to the exposed epidermal surface (1.77 cm2) of each skin disc through the openings in the upper plate of the chamber. During the 0-6 h sampling, media effluent was voided directly into empty scintillation vials over 30-min collection periods and then dissolved in scintillation cocktail and counted. At termination of sampling, the skin pieces were removed from the chamber, placed in a Petri dish, and any unabsorbed dose was removed from the skin using water-wetted swabs and placed in a scintillation vial. Rinse samples were counted and skin samples were combusted in a biological oxidizer (Biological Materials Oxidizer, R.J. Harvey Instrument Corp, Hillsdale, NJ, USA)for inclusion in the calculation of total recovery.

The amount of 14-C which penetrated skin (cumulative percent absorbed) was determined from the sum of effluent counts divided by the mean dosing solution counts. The pseudo steady-state penetration rate was computed by the plotting interval 14-C values (normalized to mass per unit surface area) vs time and then taking the slope from the linear portion of the curve. The slope values were used to caculated permeability constant (kp) values as described by Bronaugh et al., 1982.

Results and discussion

Signs and symptoms of toxicity:
no effects
Remarks:
no signs of toxicity
Dermal irritation:
no effects
Remarks:
no signs of irritation
Absorption in different matrices:
Undiluted EHD applied under occlusion did not penetrate the skin to any substantial degree, with less than 1% of the applied dose for human skin, 2-4% for rat skin and 3-6% for rabbit skin being recovered in the effluents after 6 h. In addition, 97% of the radioactivity recovered was as unabsorbed dose for human skin, with an average of 0.6% of the applied dose recovered in the effluents; this lower effluent recovery was attributed to EHD evaporation from the skin. Recovery data for animal preparations showed 94% remained on the skin surface for unabsorbed dose for rats and 85% for rabbits.

When EHD was applied as a 3% water solution to rat and human skin, effluent recovery percentages increased 2-4 times for rat skin and about 6-fold for human skin. There was also a 2-4 fold increase for rat skin and a 10-fold increase for humans in the recovery of radioactivity in combusted skin samples, indicating that skin penetration was increased in the diluted sample as compared with the undiluted sample.

Permeability constants and cumulative absorbed dose were calculated for EHD. Human skin showed the slowest penetration rate of the three species for both undiluted EHD and 3% aqueous solution.
Total recovery:
Undiluted EHD: 89.2% for human skin, 93.2% for rat and 83.3% for rabbit.
3% aqueous EHD: 78.7% for human skin, 84.2% for rat. Not assessed for rabbit.
Percutaneous absorptionopen allclose all
Dose:
100%
Parameter:
percentage
Absorption:
0.86 %
Remarks on result:
other: 6 h
Remarks:
Human, undiluted EHD
Dose:
100%
Parameter:
percentage
Absorption:
2.47 %
Remarks on result:
other: 12 h
Remarks:
Human, undiluted EHD
Dose:
100%
Parameter:
percentage
Absorption:
5.71 %
Remarks on result:
other: 24 h
Remarks:
Human, undiluted EHD
Dose:
3%
Parameter:
percentage
Absorption:
5.1 %
Remarks on result:
other: 6 h
Remarks:
Human, 3% aqueous EHD
Dose:
3%
Parameter:
percentage
Absorption:
13.94 %
Remarks on result:
other: 12 h
Remarks:
Human, 3% aqueous EHD
Dose:
3%
Parameter:
percentage
Absorption:
31.93 %
Remarks on result:
other: 24 h
Remarks:
Human, 3% aqueous EHD
Dose:
100%
Parameter:
percentage
Absorption:
3.57 %
Remarks on result:
other: 6
Remarks:
Rat, undiluted EHD
Dose:
100%
Parameter:
percentage
Absorption:
8.15 %
Remarks on result:
other: 12 h
Remarks:
Rat, undiluted EHD
Dose:
100%
Parameter:
percentage
Absorption:
17.26 %
Remarks on result:
other: 24 h
Remarks:
Rat, undiluted EHD
Dose:
3%
Parameter:
percentage
Absorption:
6.52 %
Remarks on result:
other: 6 h
Remarks:
Rat, 3% aqueous EHD
Conversion factor human vs. animal skin:
Absorption rate (mg/cm2/h), undiluted EHD, female rat compared to female human: 2.7 (0.19 rat, 0.07 human).
Cumulative dose absorbed, undiluted EHD, female rat compared to female human: 4.2 (3.57% rat, 0.86% human).

Any other information on results incl. tables

Determination of whether EHD penetrates the skin as the parent molecule or whether a metabolite is produce during penetration was investigated by quantifying the amount of undiluted EHD in the effluent liquid. More than 99% of the radioactivity from any time interval was as unmetabolised EHD. First pass metabolism in the skin itself does not play a role in the cutaneous absorption of EHD.

Evaporation as a competing factor was investigated. Approximately 25% of the undiluted EHD evaporated and was absorbed by the rat skin preparations.

Applicant's summary and conclusion

Conclusions:
In vitro skin absorption of undiluted 2-ethylhexane-1,3-diol (EHD) and 3% aqueous EHD was compared between human, rat and rabbit skin. Female rabbit skin absorbed undiluted EHD most effectively, followed by rat and lastly, human. Human skin absorbed less than 1% of the administered concentration of undiluted EHD. Absorption of undiluted EHD was approximately 3-fold higher in rats than in human skin. Absorption of dilute EHD (3% aqueous solution in water) was higher, comparable between rat and human, but half the amount absorbed by rabbit skin. No metabolism of EHD was evident in the skin or in vitro during the experiment.